Intensive research in pursuit of understanding the molecular mechanisms of insulin resistance associated with obesity and type 2 diabetes has taught us that more than one mechanism likely contributes to the final phenotype. Published and preliminary findings are consistent with the theory that plasma membrane (PM) and cytoskeletal defects are an important, unappreciated derangement in insulin resistance contributing to impaired glucose transporter GLUT4 regulation by insulin. Mechanistically, the membrane/cytoskeletal defects result from PM cholesterol accrual that is induced by physiological hyperinsulinemia, a state known to promote the progression/worsening of insulin resistance. Interestingly, removal of the excess PM cholesterol fully restores insulin sensitivity in cultured cells and skeletal muscle isolated from obese, insulin-resistant, Zucker (fa/fa) rats. New data also suggest that increased hexosamine biosynthesis pathway (HBP) activity promotes PM cholesterol accrual, membrane/cytoskeletal defects, and insulin resistance. Similar to cholesterol reduction correcting the membrane/cytoskeletal-associated insulin resistance, inhibition of the HBP blocks the membrane/cytoskeletal defects and restores insulin sensitivity. Therefore, a postulate of this application is that the HBP increases the transcriptional activity of cholesterogenic transcription factors. Also observed is that activation of AMP-activated protein kinase (AMPK), a known antidiabetic therapeutic target, is associated with a loss of PM cholesterol. The central hypothesis of the proposed research is that the breakdown of glucose homeostasis, characteristic of obesity and T2D, is secondary to PM cholesterol accrual in fat and muscle. It is also a prediction that several antidiabetic therapies improve insulin action via an unappreciated beneficial effect on cholesterol-laden, insulin-resistant PM. This will be tested in three Specific Aims. 1) Define if arrival/docking and/or fusion steps of GLUT4 translocation are compromised. Preliminary data suggest this to be a correctable distal defect. 2) Dissect the mechanism(s) by which hyperinsulinemia increases PM cholesterol. Evidence supports the idea that increased HBP activity is coupled to PM cholesterol accrual. The implications of this concept are tremendous, as other insults such as hyperglycemia and hyperlipidemia would pose the same threat and thus, accumulation of PM cholesterol may be a common mechanism by which these three consequences of insulin resistance promote the progression/worsening of the syndrome. 3) Determine if AMPK activation, exercise, and/or statins lower PM cholesterol. These results will be significant, because they are expected to provide new targets for the preventative and therapeutic interventions important to the growing numbers of insulin-resistant individuals in this country who display different biochemical signatures but a shared loss in insulin sensitivity. PUBLIC HEALTH RELEVANCE: Solving how insulin resistance develops and eventually progresses/worsens to type 2 diabetes (T2D) remains a fundamental challenge in biology and a significant issue in medicine. Our studies have discovered that the breakdown of glucose homeostasis, characteristic of obesity and T2D, is secondary to plasma membrane cholesterol accrual in fat and muscle. We have also identified means to protect against this derangement, and further investigation of the mechanisms involved will hopefully lead to new therapeutic strategies to curtail the accelerated expansion of the T2D population.